The nuclear function of the heterodimeric NF-kB transcription factor is regulated in part through reversible acetylation of its RelA subunit. We now demonstrate that the p300 and CBP acetyltransferases play a major role in the in vivo acetylation of RelA, principally targeting lysines 218, 221 and 310 for modi®cation. Analysis of the functional properties of hypoacetylated RelA mutants containing lysine-toarginine substitutions at these sites and of wild-type RelA co-expressed in the presence of a dominantly interfering mutant of p300 reveals that acetylation at lysine 221 in RelA enhances DNA binding and impairs assembly with IkBa. Conversely, acetylation of lysine 310 is required for full transcriptional activity of RelA in the absence of effects on DNA binding and IkBa assembly. Together, these ®nd-ings highlight how site-speci®c acetylation of RelA differentially regulates distinct biological activities of the NF-kB transcription factor complex.

The NF-B/Rel family of transcription factors plays a key role in regulating inflammatory and immune responses and other programs of cell growth and survival. The five known mammalian Rel genes encode seven Rel-related proteins: RelA/p65; p105 and its processing product, p50; p100 and its processing product, p52; c-Rel; and RelB. Each contains an N-terminal Rel homology domain (ϳ300 amino acids) that mediates DNA binding, dimerization, and interaction with the IB family of NF-B/Rel inhibitors. RelA, c-RelA, and RelB contain C-terminal transactivation domains, but p50 and p52 do not. Each NF-B/Rel protein forms different homo-or heterodimers with other members of the family, which may contribute to the activation of specific target genes (1, 5).The prototypical NF-B complex is a p50/RelA heterodimer. NF-B is largely sequestered in the cytoplasm through its association with an IB inhibitor. Nuclear NF-B expression is induced by various stimuli, including proinflammatory cytokines, growth factors, DNA-damaging agents, and viral proteins (13). The activation of NF-B can be divided into two phases. The first phase involves cytoplasmic events culminating in the activation of the IB kinases (IKK1 and IKK2). These kinases promote N-terminal phosphorylation of serines 32 and 36 in IB␣, leading to its polyubiquitylation and proteasome-mediated degradation. The liberated NF-B complex rapidly translocates to the nucleus, ending the first phase (13). The second phase occurs primarily in the nucleus and involves posttranslational modification of the NF-B transcription factor complex or relevant histones surrounding NF-B target genes (5). These modifications determine both the strength and duration of the NF-B-mediated transcriptional response (5).One of the nuclear events is the reversible acetylation of RelA (4). Endogenous RelA is acetylated in a stimulus-coupled manner after activation of cells with tumor necrosis factor alpha (TNF-␣), phorbol myristate acetate, or other stimuli at multiple sites, including lysines 122, 123, 218, 221, and 310 (4, 17). The acetyltransferases p300 and CBP appear to play a major role in the in vivo acetylation of RelA (6,17). Sitespecific acetylation of RelA regulates discrete biological actions of the NF-B complex (5, 6). For example, acetylation of lysine 221 by p300/CBP increases the DNA binding affinity of RelA for the B enhancer and, together with acetylation of lysine 218, impairs assembly of RelA with newly synthesized IB␣, which shuttles in and out of the nucleus. Acetylation of lysine 310 does not modulate DNA binding or IB␣ assembly but markedly enhances the transcriptional activity of NF-B. Deacetylation of lysine 310 by histone deacetylase 3 (HDAC3) or SIRT1 inhibits the transcriptional activity of RelA and augments cellular apoptosis in response to 32). While it is clear that signal-coupled acetylation of RelA participates in the nuclear regulation of NF-B action (4, 17), many unanswered questions remain. Chief among these is how the acetylation of RelA is regulated.

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Apoptosis induced by p53 has been proposed to involve activation of the transcription factor NF-B. Here we describe the novel molecular mechanism through which p53 and DNA-damaging agents activate NF-〉. NF-B induction by p53 does not occur through classical activation of the IB kinases and degradation of IB␣. Rather, p53 expression stimulates the serine/threonine kinase ribosomal S6 kinase 1 (RSK1), which in turn phosphorylates the p65 subunit of NF-B. The lower affinity of RSK1-phosphorylated p65 for its negative regulator, IB␣, decreases IB␣-mediated nuclear export of shuttling forms of NF-B, thereby promoting the binding and action of NF-B on cognate B enhancers. These findings highlight a rather unusual pathway of NF-B activation, which is utilized by the p53 tumor suppressor.